Emerging lines of evidence suggest a close link between obesity, energy metabolism, nutrients and epigenetic mechanism. Epigenetic changes, such as dynamic histone modifications, are associated with cellular metabolism and diabetic complications. Nevertheless, the molecular mechanisms mediating the crosstalk between metabolism and epigenetics remain incompletely understood. We recently discovered and comprehensively validated a new, evolutionarily-conserved lysine modification, lysine beta(?)- hydroxybutyrylation (Kbhb), on core histones. We detected 44 non-redundant Kbhb marks on histones and identified Sirt2 enzyme as the first enzyme to remove histone Kbhb. Levels of Kbhb are very dynamic and are influenced by physiological conditions (e.g., starvation and type I diabetes) and nutrition sources. Increased levels of ?-hydroxybutyrate (also called 3-hydroxybutyrate) lead to increased histone Kbhb, presumably via conversion of ?-hydroxybutyrate to ?-hydroxybutyryl CoA. Interestingly, histone Kbhb is enriched in active gene promoters, and the increased H3K9bhb levels that occur during prolonged starvation are associated with genes up-regulated in starvation-responsive metabolic pathways, thus representing a new epigenetic regulatory mark that couples metabolism to gene expression. ?-Hydroxybutyrate is a key component of ?ketone bodies? and it has been employed in dozens of anti-cancer clinical trials as a potential therapeutic in combination with other agents. The plasma/cellular concentration of ?-hydroxybutyrate can increase up to 20 mM during starvation and in pathological conditions such as diabetes mellitus (DM) and alcoholic liver damage and this can drive histone Kbhb formation. Hyperketonemia and ketoacidosis are known to increase the risk of morbidity and mortality in patients. Thus, molecular characterization of Kbhb pathway will not only improve our understanding of epigenetic mechanism but also characterize functions of ?-hydroxybutyrate in physiopathology. We hypothesize that the Kbhb pathway is molecularly distinct from the lysine acetylation pathway. We therefore propose to characterize the Kbhb pathway by defining its key regulatory elements, including its substrates, a unique set of regulatory enzymes and direct binding proteins, thus laying a foundation for studying its biology functions. We will use an integrated strategy in this study involving enzymology, chemical biology, biochemistry and proteomics approaches. Our team, the Zhao laboratory and the Cole laboratory, is well positioned to carry out this project, because of our combined expertise in these areas and the relevant preliminary data that we have already obtained. In this proposal, we will first comprehensively identify and quantify dynamic changes of Kbhb-containing substrates using a quantitative proteomics approach. We will then identify and characterize Kbhb-regulatory enzymes that can add or remove Kbhb. We will finally identify and confirm the direct protein binders of histone Kbhb peptides.